霞水母中胶原蛋白的分离及生理活性初探
摘要
霞水母是严重威胁我国渤海和黄海北部的渔业生态资源的一种海洋生态灾害物种,霞水母爆发与赤潮并列为我国海洋生态的两大灾难。为了解决霞水母爆发对我国海洋生态环境的灾难性影响,急需进行霞水母的资源化利用方面的研究,以便增加其附加值,提高渔民的捕捞积极性,确保霞水母的捕捞死亡率大于其自然增长率,逐渐消除其对海洋生态环境的影响。然而,迄今为止,有关霞水母的资源化利用方面的研究尚属空白,国内外迄今未见任何相关报道。
胶原蛋白作为细胞外基质中起支持作用的主要成分,具有“Gly-X-Y”的特异性氨基酸组成和螺旋结构,因其还具有抗原性低、可生物降解性强、生物相容性好等优点,故在生物学、医学、食品科学和美容业等领域中具有巨大的研究开发潜力和广阔的应用前景。
国内外对胶原蛋白的分离制备主要以哺乳动物皮肤、软骨以及鱼类皮肤等组织为原料,对其研究主要集中在分离制备和生物材料领域的开发应用方面。到目前为止,对腔肠动物胶原蛋白的研究尚较少,霞水母来源胶原蛋白的分离纯化及其生理活性方面的研究迄今国内外未见任何报道。
为了实现霞水母的资源化开发利用,本文在国内外首次就霞水母中含有的胶原蛋白进行了研究,建立了霞水母胶原蛋白的分离制备和纯化方法,并就其是否对类风湿性关节炎的预防和治疗具有生物活性进行了探索性研究,为将霞水母中所含胶原蛋白作为天然海洋胶原蛋白资源加以开发利用,运用环境经济理念,消除这一海洋生态灾难,提供了工作基础和实验依据。
本文用酶法从霞水母中提取得到了胶原蛋白。根据聚丙烯酰胺凝胶电泳、变性聚丙烯酰胺凝胶电泳图谱推测霞水母胶原蛋白可能是由三条分子大小在66.2 KDa-116 KDa之间不同亚基组成的三聚体。样品中蛋白质含量为98.0%。本实验所测得胶原蛋白的羟脯氨酸含量为10.54%,而糖无检出,表明胶原蛋白纯度较高。
急性毒性试验表明,在剂量为15000 mg/kg霞水母胶原蛋白的情况下,未出现小鼠死亡,也未有明显毒性反应。
本实验对霞水母胶原蛋白口服治疗大鼠佐剂型关节炎进行了初步探索。发现口服霞水母胶原蛋白可有效减轻AA大鼠的关节肿胀度,显著降低AA大鼠血清MDA、NO水平,提高血清SOD的活性。通过这些指标,可以推测胶原蛋白对大鼠佐剂型关节炎可能有一定的治疗效果。
Cyanea nozakii is a kind of harmful jellyfish for marine ecosystem which bloom out in the Bohai Sea and Northern Yellow Sea of China. In fact, the jellyfish bloom and Harmful Algal Bloom are top two ecological disaster of Chinese marine ecosystem, threatening the fishery industry. In order to eliminate threaten of jellyfish bloom on the marine fishery industry of our country. It is desiderated to undertake the basic research about how to use it as a kind of new marine resource. From the environmental economic point of view, it is the best solution try to find the way to utilize it. About how to use it as a kind of resource, however, we know nothing. Nothing has been reported about the basic knowledge and information about basic components, as well as their function up to now.
Collagen is a major component of extracellular matrix (ECM), and has a unique three-dimensional structure with a unique amino acid composition. Owing to wonderful properties of its low-antigenicity, easy-biodegradability and excellent biocompatibility, collagen is used more and more widely in areas of biological material development, food industry, cosmetic manufacture, and medical production as well.
Recently, there have been many interests in establishing efficient techniques to isolate collagen from skin of fish, and skin, cartilage and soon from mammals, and to apply them in development of various kinds of biological materials. However, there has been no report about the collagen from the Cyanea nozakii, as well as its biological functions on adjuvant arthritis treatment.
For the first time, the collagen from Cyanea nozakii has been investigated comprehensively. The isolation and purification methods have been established, and the potential physiologic activity of the collagen from Cyanea nozakii on the prevention and treating of adjuvant arthritis has also been explored. The data and results collected and recorded on this thesis can be used as the basic information for the further research and development of Cyanea nozakii, as a kind of new marine conllagen resources.
The collagen from Cyanea nozakii has been extracted by enzymatic method. The collagen sample obtained was examined by PAGE and SDS-PAGE, which indicate that collagen from Cyanea nozakii may have the chain composition of a heterotrimer and the subunits weighed from 66.2 KDa to 116 KDa. Respectively, the percentage of hydroxyl-proline in the collagen after purification was 10.54%, the concentration of the protein was 98.0%, and the concentration of the polysaccharide was no detection.
The acute toxicity experiment showed that injecting big dose, mice didn’t die and hadn’t obvious toxicity reaction. In a word, the collagen from Cyanea nozakii has no toxicity, which security dose was 15000 mg/kg.
In this study, therapeutic effect of Cyanea nozakii collagen by oral administration on adjuvant arthritis in rats was initiatory explored. Cyanea nozakii collagen at different concentration could ameliorate the adjuvant induced arthritis, suppress the concentrations of NO and MDA in the blood serum and increase the activity of SOD in the blood serum. From these indexes can presume that, Cyanea nozakii collagen is probably ameliorating the adjuvant induced arthritis.
胶原蛋白作为细胞外基质中起支持作用的主要成分,具有“Gly-X-Y”的特异性氨基酸组成和螺旋结构,因其还具有抗原性低、可生物降解性强、生物相容性好等优点,故在生物学、医学、食品科学和美容业等领域中具有巨大的研究开发潜力和广阔的应用前景。
国内外对胶原蛋白的分离制备主要以哺乳动物皮肤、软骨以及鱼类皮肤等组织为原料,对其研究主要集中在分离制备和生物材料领域的开发应用方面。到目前为止,对腔肠动物胶原蛋白的研究尚较少,霞水母来源胶原蛋白的分离纯化及其生理活性方面的研究迄今国内外未见任何报道。
为了实现霞水母的资源化开发利用,本文在国内外首次就霞水母中含有的胶原蛋白进行了研究,建立了霞水母胶原蛋白的分离制备和纯化方法,并就其是否对类风湿性关节炎的预防和治疗具有生物活性进行了探索性研究,为将霞水母中所含胶原蛋白作为天然海洋胶原蛋白资源加以开发利用,运用环境经济理念,消除这一海洋生态灾难,提供了工作基础和实验依据。
本文用酶法从霞水母中提取得到了胶原蛋白。根据聚丙烯酰胺凝胶电泳、变性聚丙烯酰胺凝胶电泳图谱推测霞水母胶原蛋白可能是由三条分子大小在66.2 KDa-116 KDa之间不同亚基组成的三聚体。样品中蛋白质含量为98.0%。本实验所测得胶原蛋白的羟脯氨酸含量为10.54%,而糖无检出,表明胶原蛋白纯度较高。
急性毒性试验表明,在剂量为15000 mg/kg霞水母胶原蛋白的情况下,未出现小鼠死亡,也未有明显毒性反应。
本实验对霞水母胶原蛋白口服治疗大鼠佐剂型关节炎进行了初步探索。发现口服霞水母胶原蛋白可有效减轻AA大鼠的关节肿胀度,显著降低AA大鼠血清MDA、NO水平,提高血清SOD的活性。通过这些指标,可以推测胶原蛋白对大鼠佐剂型关节炎可能有一定的治疗效果。
Cyanea nozakii is a kind of harmful jellyfish for marine ecosystem which bloom out in the Bohai Sea and Northern Yellow Sea of China. In fact, the jellyfish bloom and Harmful Algal Bloom are top two ecological disaster of Chinese marine ecosystem, threatening the fishery industry. In order to eliminate threaten of jellyfish bloom on the marine fishery industry of our country. It is desiderated to undertake the basic research about how to use it as a kind of new marine resource. From the environmental economic point of view, it is the best solution try to find the way to utilize it. About how to use it as a kind of resource, however, we know nothing. Nothing has been reported about the basic knowledge and information about basic components, as well as their function up to now.
Collagen is a major component of extracellular matrix (ECM), and has a unique three-dimensional structure with a unique amino acid composition. Owing to wonderful properties of its low-antigenicity, easy-biodegradability and excellent biocompatibility, collagen is used more and more widely in areas of biological material development, food industry, cosmetic manufacture, and medical production as well.
Recently, there have been many interests in establishing efficient techniques to isolate collagen from skin of fish, and skin, cartilage and soon from mammals, and to apply them in development of various kinds of biological materials. However, there has been no report about the collagen from the Cyanea nozakii, as well as its biological functions on adjuvant arthritis treatment.
For the first time, the collagen from Cyanea nozakii has been investigated comprehensively. The isolation and purification methods have been established, and the potential physiologic activity of the collagen from Cyanea nozakii on the prevention and treating of adjuvant arthritis has also been explored. The data and results collected and recorded on this thesis can be used as the basic information for the further research and development of Cyanea nozakii, as a kind of new marine conllagen resources.
The collagen from Cyanea nozakii has been extracted by enzymatic method. The collagen sample obtained was examined by PAGE and SDS-PAGE, which indicate that collagen from Cyanea nozakii may have the chain composition of a heterotrimer and the subunits weighed from 66.2 KDa to 116 KDa. Respectively, the percentage of hydroxyl-proline in the collagen after purification was 10.54%, the concentration of the protein was 98.0%, and the concentration of the polysaccharide was no detection.
The acute toxicity experiment showed that injecting big dose, mice didn’t die and hadn’t obvious toxicity reaction. In a word, the collagen from Cyanea nozakii has no toxicity, which security dose was 15000 mg/kg.
In this study, therapeutic effect of Cyanea nozakii collagen by oral administration on adjuvant arthritis in rats was initiatory explored. Cyanea nozakii collagen at different concentration could ameliorate the adjuvant induced arthritis, suppress the concentrations of NO and MDA in the blood serum and increase the activity of SOD in the blood serum. From these indexes can presume that, Cyanea nozakii collagen is probably ameliorating the adjuvant induced arthritis.
引文
1.仲霞铭,汤建华,刘培延.霞水母暴发与海洋生态之关联性探讨[J].现代渔业信息,2004,19(3):15—17
2.卢振彬,戴泉水,颜尤明.福建东山岛海域霞水母的渔业生物学研究[J].应用生态学报,2003,14(6):973—97
3.彭永章,王惠瑛.霞水母资源亟待开发研究-浅论渔船捕捞与机械加工霞水母一体化技术的开发[J].中国渔业经济,2007,2:43—45
4.葛利君,何德民.生态危机的标志性信号[J].中国水产,2004,9:23—28
5.景沛.胶原蛋白质(Ⅰ~ⅩⅧ)[M].生命化学,1995,15(6):30—31
6.韩贻仁,樊廷俊,杨晓梅,等.分子细胞生物学第二版[M].北京科学出版社,2002.154—155
7.王唯玮.海星的生理、药理活性物质[J].中国海洋药物,1986,5(4):46—57
8.任俊莉,付丽红,邱化玉.胶原蛋白的应用及其发展前景[J].中国皮革,2003,32(23):16—17
9.顾其胜,侯春林.天然可降解性生物医学材料的研究进度[J].上海生物医学工程,1999,20(1):51—55
10.林伟,穆畅通.胶原有关的化学研究进展[J].化学进展,2000,12(2):218—227
11.永井裕,藤本大三郎.胶原蛋白实验方法[M].上海中医学院出版社,1992.4—8
12.胡胜,李志强,陈敏.皮胶原蛋白的酶法提取及在高附加值领域的应用[J].皮革科学与工程,2002,12(5):38—43
13.王碧,贾冬英,张铭让,等.色谱法在胶原蛋白分离分析中的应用[J].化学通报,2001,65(1):1—6
14.鲁元刚.胶原在皮肤组织工程中的应用[J].重庆医学,2003,32(5):609—612
15.任俊莉,付丽红,邱化玉,等.胶原蛋白的应用及其发展前景[J].中国皮革,2003,32(23):16—17
16.顾汉卿,徐国风.生物医学工程学丛书之二——生物医学材料学第一版[M].天津科技翻译出版公司,1993,324—325
17.武继民,叶萍,孙伟健,等.胶原海绵及其止血性能的研究[J].生物医学工程学杂志,1998,15(1):63—65
18. Bader A,Steinhoff G,Strobl K,et al. Engineering of human vascular aortic tissue based ona xenogeneic starter matrix[J].Transplantation,2000,70:7—14
19. Wilson G J, Courtman D W, Klement P,et al. A cellular matrix:a bio-materials approach for coronary artery bypass an heart valve replacement[J].AnnThoracSur g,1995,60:353—358
20.但卫华,曾睿.生物质与生物质工程[J].中国皮革,2002,31(11):31—35
21.刘慧玲,王栋,章金刚.胶原蛋白在临床医学中的应用[J].北京生物医学工程,2005,24(3):239—241
22.朱梅湘,穆畅道,陈武勇,等.胶原基生物膜材料的制备与应用[J].中国皮革,2003,7:161—164
23. Huynh T,Abraham G,Murray J,et al.Remodeling of an acellular collagen graft into a physiologically responsive neovessel[J].Nat Biotechnol,1999,17(11):1083—1086
24. Ignatius A,Blessing H,Liedert A,et al.Tissue engineering of bone:effects of mechanical strain on osteoblastic cells in type I collagen matrices[J].Biomaterials,2005, 26:311—318
25. Hobden J A,Reidy J J O’callaghan R J,et al. Treatment of experimental pseudomonas kerat it is using collagen shields containing tobrmycin [J]. Arc ho ph thal mol ,198 8,106:1605—1607
26.陈悦玲,甄兆忠,何志远.胶原膜在角膜手术中应用的初步报告[J].中国实用眼科杂志,1996,14:313—314
27.王越.角膜胶原膜的研究进展[J].眼科杂志,2004,13(6):367—371
28.赵世红,王滨生,侯勤英.角膜胶原膜的研制及临床应用[J].中国实用眼科杂志,1998,16(12):731—732
29.周文天,俞益丰,邓青,等.FK506胶原药膜抑制兔角膜移植的免疫排斥反应[J].眼科新进展,2004,24(4):254—256
30. Teebken C E,Bader A,Steinhoff G,et al.Tissue engineering of vascular grafts:human cell seeding of decellularised porcine matrix[J].Eur JVasc Endovasc Surg,2000,19:381—386
31.裘炳毅.生物技术制剂及其在化妆品的应用[J].日用化学工业,1996,1:52—56
32.王远亮,魏小姬.胶原和明胶在化妆品中的应用[J].明胶科学与技术,1987,7(3):103—112
33.朱铁成.胶原蛋白在系列化妆品中的应用[J].北京日化,2000,4:18—21
34.胡胜,李志强,陈敏.猪皮胶原的酶法提取[J].中国皮革,2003,32(5):6—10
35.李开雄,赵志远,刘霞.猪皮中胶原蛋白的提取及其应用[J].肉类研究,1994,4:43—47
36.刘白玲.胶原在生物医学领域的应用[J].皮革科学与工程,1999,9(3):35—42
37.唐传核,彭志英.胶原的开发及利用[J].肉类研究,2000,3:41—43
38.王霞,魏大鹏,蔡美英,等.口服鸡Ⅱ型胶原治疗大鼠的佐剂型关节炎[J].华西药学杂志. 2003,18(4) :255—257
39. Jacobsonpb,borgansj,wilcoxdm,et al.A new spin on an old model:in vim evaluation of disease progress by magnetic resonance imaging with respect to standard inflammatory parameters and stopathology inadiuvant arthritic rat[J].Anhritis Rheum,1999,42(10):2060—2073
40. Tian J,Olclyia,Hanssenl,et al.Antigenbased immunotherapy for autoimmune disease:from animal models to human[J].Immunol Today,1999,20(4):190—195
41. Trenthamde,Dynesius T A,Oraave J,et al.Effects of oral administration of typeII collagen on rheumatoid arthritis[J].Science,1993,261:1727—1730
42. Chen Y,Kuchrkv K,Inobe J,et al.Regulatory T cell clones induced by oral tolerance:suppression of autoimmune[J].Science,1994,265:1237—1240
43. Weiner H L.Oral tolerance:immune mechanisms and treatment of autoimmune diseases[J].Immunol Today,1997.18:335—343
44.刘庆慧,王彩理,刘丛力.鱼鳞胶原蛋白研究[J].海洋水产研究,2000,21(3):57—60
45.唐传核,彭志荚.一种新型功能性食品基料——胶原多肽[J].现代商贸工业,2001(5):44—45
46. Li H,Liu B,Gao L.Z.et al.Studies on bullfrog skin collagen[J].Food Chem,2004,84:65-69
47. Kittiphattanabawon P, Benjakuls, Viscsanguan W, et al. Charaeterisafion of acid-soluble collagen from skin and bone of big eyesnapper (Priacanthus tayenus)[J]. Food chemistry, 2005(89):363—372
48.顾其胜,蒋丽霞.胶原蛋白与临床医学[M].第二军医大学出版社,2003
49. Pace J M,Coado M,Missero C,et al.Identification, characterization and expression analysis of a new fibrillar collagen gene [J]. Matrix Biology,2003,22(1):3—14
50. Yata M, Yoshida C, Fujisawa S,et al.Identifcation and characterization of molecular species of collagen in fish skin [J]. Food Sci,2001,66(2):247—251
51. Takeshi N,Yoko A,Nobutaka S.Collagen of the skin of ocellate pufer fish (Takifugu rubripes)[J].Food Chem,2002,78:173—177
52. Rigo C,Hartmann D J,Bairati A.Electrophoretic and immunochemical study of collagens from sepia officinalis cartilage[J].Biochim Biophy Acta,2002,1572:77—84
53. Takeshi N,Nobutaka S.Isolation of collagen from fish waste material-skin,bone and fins[J].Food Chem,000,68:277—281
54.秦玉青,刘承初,王懂.鱿鱼皮胶原蛋白的测定与回收[J].上海水产大学学报,2002,11(2):138—144
55.刘庆慧,王彩理,刘丛力.鱼鳞胶原蛋白研究[J].海洋水产研究,2000,21(3):57—61
56.赫林华,李八方.多棘海盘车体壁胶原蛋白的研究[J].中国水产科学,1999,6(6):18—21
57.郭瑶,曾名勇,崔文萱.水产胶原蛋白及胶原多肽的研究进展[J].水产科学,2006年2月第25卷第2期,101—104
58.陈胜军,曾名勇,董士远.水产胶原蛋白及其活性肽的研究进展[J].水产科学,2004,23(6):44—-46
59.张文熊,李欣,魏娜.酶法提取胶原的研究[J],中国皮革,2006,35(23),15—17
60.蒋挺大,张春萍.胶原蛋白[M].化学工业出版社, 2001.1
61. Kimura S, Omura Y, Ishida M, et al.Molecular Characterization of Fibrillar Collagen From the Body Wall of Starfish Asterias Amurensis[J].Comparative Biochemical Physiology,1993,104B(4):663—668
62.李国英,张忠楷,雷苏,等.胶原、明胶和水解胶原蛋白的性能差异[J].四川大学学报,2005,37(4):54—37
63.金勇,徐社阳,刘宗惠,等.猪皮提取胶原的研究[J].精细化工,2001,18(5):302—304
64.陈柏松,徐玉,钟淑琦,等.大鼠佐剂性关节炎模型的建立与评价[J].哈尔滨医科大学学报,2005,39( 6):489—191
65.张志胜,刘安军,刘媛,等.口服羊软骨II型胶原蛋白抑制小鼠佐剂性关节炎研究[J].河北农业大学学报,2006,29(5):76—80
66. Ping Zhu, Xiao-Yan Li, Hong-Kun Wang,et al.Oral administration of type-II collagen peptide 250--270 suppresses specific cellular and humoral immune response in collagen-induced arthritis[J].Clinical Immunology 2007,122: 75—84
67.钱娴娟,赵文明,胡永秀,等.口服Ⅱ型胶原蛋白诱导佐剂性关节炎大鼠免疫耐受的研究[J].中华风湿病学杂志2002, 6(5),331—334
68.杜显光,卜宗式.竹荪深层发酵产物的毒性及降血脂作用分析[J].中国食用菌,1997,16(3):27—30
69.朱玉强.刺参多糖急性毒理研究及其安全性评价[J].四川食品与发酵,2005,3(41):48—50
70. Nagy G, Clark J M, Buzás E I,et al.Nitric oxide,chronic inflammation and autoimmunity[J].Immunol Lett. 2007,111(1):1—5
71. Young-Gyu Cho,Mi-La Cho,So-Youn Min,et al.Type II collagen autoimmunity in a mouse model of human rheumatoid arthritis[J]. Autoimmunity Reviews 2007,7:65—70
72. Weber; Jon, Use of jellyfish collagen (type II) in the treatment of rheumatoid arthritis[P].United States. Patent.6894029. 2005—05—17
73. Biji T,Kurien,K H Michael B,et al,Oxidatively modified autoantigens in autoimmune diseases[J].Free Radical Biology & Medicine,2006,41:549—556
74. Elisabeth M,Birgit M,Reipert D,et al.Induction of immune tolerance by oral IVIG[J]. International Immunopharmacology, 2007,7:351—359
2.卢振彬,戴泉水,颜尤明.福建东山岛海域霞水母的渔业生物学研究[J].应用生态学报,2003,14(6):973—97
3.彭永章,王惠瑛.霞水母资源亟待开发研究-浅论渔船捕捞与机械加工霞水母一体化技术的开发[J].中国渔业经济,2007,2:43—45
4.葛利君,何德民.生态危机的标志性信号[J].中国水产,2004,9:23—28
5.景沛.胶原蛋白质(Ⅰ~ⅩⅧ)[M].生命化学,1995,15(6):30—31
6.韩贻仁,樊廷俊,杨晓梅,等.分子细胞生物学第二版[M].北京科学出版社,2002.154—155
7.王唯玮.海星的生理、药理活性物质[J].中国海洋药物,1986,5(4):46—57
8.任俊莉,付丽红,邱化玉.胶原蛋白的应用及其发展前景[J].中国皮革,2003,32(23):16—17
9.顾其胜,侯春林.天然可降解性生物医学材料的研究进度[J].上海生物医学工程,1999,20(1):51—55
10.林伟,穆畅通.胶原有关的化学研究进展[J].化学进展,2000,12(2):218—227
11.永井裕,藤本大三郎.胶原蛋白实验方法[M].上海中医学院出版社,1992.4—8
12.胡胜,李志强,陈敏.皮胶原蛋白的酶法提取及在高附加值领域的应用[J].皮革科学与工程,2002,12(5):38—43
13.王碧,贾冬英,张铭让,等.色谱法在胶原蛋白分离分析中的应用[J].化学通报,2001,65(1):1—6
14.鲁元刚.胶原在皮肤组织工程中的应用[J].重庆医学,2003,32(5):609—612
15.任俊莉,付丽红,邱化玉,等.胶原蛋白的应用及其发展前景[J].中国皮革,2003,32(23):16—17
16.顾汉卿,徐国风.生物医学工程学丛书之二——生物医学材料学第一版[M].天津科技翻译出版公司,1993,324—325
17.武继民,叶萍,孙伟健,等.胶原海绵及其止血性能的研究[J].生物医学工程学杂志,1998,15(1):63—65
18. Bader A,Steinhoff G,Strobl K,et al. Engineering of human vascular aortic tissue based ona xenogeneic starter matrix[J].Transplantation,2000,70:7—14
19. Wilson G J, Courtman D W, Klement P,et al. A cellular matrix:a bio-materials approach for coronary artery bypass an heart valve replacement[J].AnnThoracSur g,1995,60:353—358
20.但卫华,曾睿.生物质与生物质工程[J].中国皮革,2002,31(11):31—35
21.刘慧玲,王栋,章金刚.胶原蛋白在临床医学中的应用[J].北京生物医学工程,2005,24(3):239—241
22.朱梅湘,穆畅道,陈武勇,等.胶原基生物膜材料的制备与应用[J].中国皮革,2003,7:161—164
23. Huynh T,Abraham G,Murray J,et al.Remodeling of an acellular collagen graft into a physiologically responsive neovessel[J].Nat Biotechnol,1999,17(11):1083—1086
24. Ignatius A,Blessing H,Liedert A,et al.Tissue engineering of bone:effects of mechanical strain on osteoblastic cells in type I collagen matrices[J].Biomaterials,2005, 26:311—318
25. Hobden J A,Reidy J J O’callaghan R J,et al. Treatment of experimental pseudomonas kerat it is using collagen shields containing tobrmycin [J]. Arc ho ph thal mol ,198 8,106:1605—1607
26.陈悦玲,甄兆忠,何志远.胶原膜在角膜手术中应用的初步报告[J].中国实用眼科杂志,1996,14:313—314
27.王越.角膜胶原膜的研究进展[J].眼科杂志,2004,13(6):367—371
28.赵世红,王滨生,侯勤英.角膜胶原膜的研制及临床应用[J].中国实用眼科杂志,1998,16(12):731—732
29.周文天,俞益丰,邓青,等.FK506胶原药膜抑制兔角膜移植的免疫排斥反应[J].眼科新进展,2004,24(4):254—256
30. Teebken C E,Bader A,Steinhoff G,et al.Tissue engineering of vascular grafts:human cell seeding of decellularised porcine matrix[J].Eur JVasc Endovasc Surg,2000,19:381—386
31.裘炳毅.生物技术制剂及其在化妆品的应用[J].日用化学工业,1996,1:52—56
32.王远亮,魏小姬.胶原和明胶在化妆品中的应用[J].明胶科学与技术,1987,7(3):103—112
33.朱铁成.胶原蛋白在系列化妆品中的应用[J].北京日化,2000,4:18—21
34.胡胜,李志强,陈敏.猪皮胶原的酶法提取[J].中国皮革,2003,32(5):6—10
35.李开雄,赵志远,刘霞.猪皮中胶原蛋白的提取及其应用[J].肉类研究,1994,4:43—47
36.刘白玲.胶原在生物医学领域的应用[J].皮革科学与工程,1999,9(3):35—42
37.唐传核,彭志英.胶原的开发及利用[J].肉类研究,2000,3:41—43
38.王霞,魏大鹏,蔡美英,等.口服鸡Ⅱ型胶原治疗大鼠的佐剂型关节炎[J].华西药学杂志. 2003,18(4) :255—257
39. Jacobsonpb,borgansj,wilcoxdm,et al.A new spin on an old model:in vim evaluation of disease progress by magnetic resonance imaging with respect to standard inflammatory parameters and stopathology inadiuvant arthritic rat[J].Anhritis Rheum,1999,42(10):2060—2073
40. Tian J,Olclyia,Hanssenl,et al.Antigenbased immunotherapy for autoimmune disease:from animal models to human[J].Immunol Today,1999,20(4):190—195
41. Trenthamde,Dynesius T A,Oraave J,et al.Effects of oral administration of typeII collagen on rheumatoid arthritis[J].Science,1993,261:1727—1730
42. Chen Y,Kuchrkv K,Inobe J,et al.Regulatory T cell clones induced by oral tolerance:suppression of autoimmune[J].Science,1994,265:1237—1240
43. Weiner H L.Oral tolerance:immune mechanisms and treatment of autoimmune diseases[J].Immunol Today,1997.18:335—343
44.刘庆慧,王彩理,刘丛力.鱼鳞胶原蛋白研究[J].海洋水产研究,2000,21(3):57—60
45.唐传核,彭志荚.一种新型功能性食品基料——胶原多肽[J].现代商贸工业,2001(5):44—45
46. Li H,Liu B,Gao L.Z.et al.Studies on bullfrog skin collagen[J].Food Chem,2004,84:65-69
47. Kittiphattanabawon P, Benjakuls, Viscsanguan W, et al. Charaeterisafion of acid-soluble collagen from skin and bone of big eyesnapper (Priacanthus tayenus)[J]. Food chemistry, 2005(89):363—372
48.顾其胜,蒋丽霞.胶原蛋白与临床医学[M].第二军医大学出版社,2003
49. Pace J M,Coado M,Missero C,et al.Identification, characterization and expression analysis of a new fibrillar collagen gene [J]. Matrix Biology,2003,22(1):3—14
50. Yata M, Yoshida C, Fujisawa S,et al.Identifcation and characterization of molecular species of collagen in fish skin [J]. Food Sci,2001,66(2):247—251
51. Takeshi N,Yoko A,Nobutaka S.Collagen of the skin of ocellate pufer fish (Takifugu rubripes)[J].Food Chem,2002,78:173—177
52. Rigo C,Hartmann D J,Bairati A.Electrophoretic and immunochemical study of collagens from sepia officinalis cartilage[J].Biochim Biophy Acta,2002,1572:77—84
53. Takeshi N,Nobutaka S.Isolation of collagen from fish waste material-skin,bone and fins[J].Food Chem,000,68:277—281
54.秦玉青,刘承初,王懂.鱿鱼皮胶原蛋白的测定与回收[J].上海水产大学学报,2002,11(2):138—144
55.刘庆慧,王彩理,刘丛力.鱼鳞胶原蛋白研究[J].海洋水产研究,2000,21(3):57—61
56.赫林华,李八方.多棘海盘车体壁胶原蛋白的研究[J].中国水产科学,1999,6(6):18—21
57.郭瑶,曾名勇,崔文萱.水产胶原蛋白及胶原多肽的研究进展[J].水产科学,2006年2月第25卷第2期,101—104
58.陈胜军,曾名勇,董士远.水产胶原蛋白及其活性肽的研究进展[J].水产科学,2004,23(6):44—-46
59.张文熊,李欣,魏娜.酶法提取胶原的研究[J],中国皮革,2006,35(23),15—17
60.蒋挺大,张春萍.胶原蛋白[M].化学工业出版社, 2001.1
61. Kimura S, Omura Y, Ishida M, et al.Molecular Characterization of Fibrillar Collagen From the Body Wall of Starfish Asterias Amurensis[J].Comparative Biochemical Physiology,1993,104B(4):663—668
62.李国英,张忠楷,雷苏,等.胶原、明胶和水解胶原蛋白的性能差异[J].四川大学学报,2005,37(4):54—37
63.金勇,徐社阳,刘宗惠,等.猪皮提取胶原的研究[J].精细化工,2001,18(5):302—304
64.陈柏松,徐玉,钟淑琦,等.大鼠佐剂性关节炎模型的建立与评价[J].哈尔滨医科大学学报,2005,39( 6):489—191
65.张志胜,刘安军,刘媛,等.口服羊软骨II型胶原蛋白抑制小鼠佐剂性关节炎研究[J].河北农业大学学报,2006,29(5):76—80
66. Ping Zhu, Xiao-Yan Li, Hong-Kun Wang,et al.Oral administration of type-II collagen peptide 250--270 suppresses specific cellular and humoral immune response in collagen-induced arthritis[J].Clinical Immunology 2007,122: 75—84
67.钱娴娟,赵文明,胡永秀,等.口服Ⅱ型胶原蛋白诱导佐剂性关节炎大鼠免疫耐受的研究[J].中华风湿病学杂志2002, 6(5),331—334
68.杜显光,卜宗式.竹荪深层发酵产物的毒性及降血脂作用分析[J].中国食用菌,1997,16(3):27—30
69.朱玉强.刺参多糖急性毒理研究及其安全性评价[J].四川食品与发酵,2005,3(41):48—50
70. Nagy G, Clark J M, Buzás E I,et al.Nitric oxide,chronic inflammation and autoimmunity[J].Immunol Lett. 2007,111(1):1—5
71. Young-Gyu Cho,Mi-La Cho,So-Youn Min,et al.Type II collagen autoimmunity in a mouse model of human rheumatoid arthritis[J]. Autoimmunity Reviews 2007,7:65—70
72. Weber; Jon, Use of jellyfish collagen (type II) in the treatment of rheumatoid arthritis[P].United States. Patent.6894029. 2005—05—17
73. Biji T,Kurien,K H Michael B,et al,Oxidatively modified autoantigens in autoimmune diseases[J].Free Radical Biology & Medicine,2006,41:549—556
74. Elisabeth M,Birgit M,Reipert D,et al.Induction of immune tolerance by oral IVIG[J]. International Immunopharmacology, 2007,7:351—359